mehari biol 3200-exam 2

essential nutrients

nutrients that must be provided to an organism

macronutrients

nutrients required in large quantities

major elements in macromolecules-C, O, H, N, P, S

ions necessary for protein function-Mg2+, Ca2+, Fe2+, K+

micronutrients

nutrients required in small quantities

trace elements necessary for enzyme function-Co, Cu, Mn, Zn, Mo, Ni

heterotrophs

organisms that use preformed organic molecules and release CO2

autotrophs

organisms that fix CO2 and assemble it into organic molecules (mostly sugars)

phototrophs and lithotrophy

phototrophy

organisms that use light as energy source

lithotrophy

organisms that get energy from oxidation of minerals

what happens if autotrophs outgrow heterotrophs

make excess organic carbon and will eventually run out of CO2

passive transport

movement of substances across cell membrane without requiring energy

simple and facilitated diffusion

simple diffusion

movement of small, uncharged molecules straight through membrane

facilitated diffusion

movement of large or charged molecules across membrane through a transport protein

solutes move [high]->[low]

active transport

movement of substances across cell membrane using energy

coupled transport and ABC transport

coupled transport

movement of a driving ion down its gradient to help move a solute up its gradient

symport and antiport

symport

coupled transport in which the 2 molecules travel in the same direction

ex/ lactose symporter

antiport

coupled transport in which actively transported molecule and driving ion move in opposite directions

ex/ Na+/H+ antiporter

ATP-binding cassette superfamily (ABC transporter)

found in all 3 domains of life

uptake and efflux pumps

uptake ABC transportes

transport nutrients

1. solute binds to binding protein and complex binds to membrane transporter

2. ATPase activity of 1 component powers opening of channel

3. solute moves into cell

efflux ABC transporters

multidrug efflux pumps; multi-drug resistance

binary fission

bacterial cell division

1 parent splits into 2 identical daughter cells

growth rate

(exponential) rate of increase in cell numbers or biomass

proportional to population size at a given time

number of cells from binary fission

2^n where n=number of generations

limiting factor

environmental factor that prevents a population from increasing infinitely

batch culture

a liquid medium within a closed system

growth curve phases

lag, log, stationary, death

lag phase

net increase in cells=0

cells prepare for growth

log phase

exponential, continuous cell growth

stationary phase

cells stop growing and turn on stress response to retain viability

death phase

cels die with "half-life" similar to radioactive decay

continuous culture

all cells in population achieve a steady state

allows detailed study of bacterial physiology

chemostat

system that ensures logarithmic growth by continuously adding and removing equal amounts of culture media

generation time

time it takes for a population to double

G=t x n

final number of cells after undergoing binary fission

Nt=N0 x 2^n

number of generations

n=3.3log(Nt/N0)

liquid or broth culture media

media useful for studying the growth characteristics of pure culture

solid (gelled w/ agar) culture media

media useful to separate mixed cultures

able to see color and morphology

types of media

complex, synthetic, enriched, selective, and differential

complex media

nutrient rich but poorly defined media

synthetic media

precisely defined media

enriched media

complex media to which specific blood components are added

selective media

favor growth of one organism over another

differential media

exploit differences between 2 species that grow equally well

macconkey agar

selective and differential media that selects for gram - bacteria and differentiates them based on ability to metabolize lactose

contains lactose, peptones, and neutral red indicator

ways to isolate pure colonies

dilution streaking and spread plate

dilution streaking

dragging a loop across the surface of agar plate

spread plate

serial dilutions performed on liquid culture

small amount of each is dilution plated and plate with isolated colonies is used

why count bacteria

helps determine infectious dose and determine actual infection vs contamination of specimens

direct microscope count

count live and dead cells on special microscope slide (counting chamber)

fluorescence-activated cell sorter (FACS)

fluorescent cells passed through small opening then past a laser

detector measure size scatter--measure particle size and shape

optical density (spectrophotometer)

fastest way to measure cell density

viable cell count

count replicating and colony forming cells via pour plate method

pour plate method

serial dilutions performed on liquid culture

small amount of each dilution is then plated

plate with 30-300 colony forming units (CFU) is counted

CFU/mL

number of colonies x dilution factor / volume transferred

how to determine dilution factor

volume transferred + volume of broth in tube

catabolism

breakdown of complex molecules into simpler ones

anabolism

metabolic pathways that construct molecules

required energy provided by catabolism

ways to transport energy in cell

in form of electrons or energy carriers

electron carriers

molecules that gain or release small amounts of energy in reversible reactions and can transfer electrons

ex/ NADH, FADH2, ATP

nicotinamide adenine dinucleotide (NADH)

electron carrier that carries 2 or 3x as much energy as ATP

NAD+

oxidized form of NADH

accepts 2H+ and 2e- in reduction reaction to form NADH

flavin adenine dinucleotide (FAD)

coenzyme that can transfer electrons

reduced by 2e- and 2 protons

FADH2

reduced form of FAD

adenosine triphospate (ATP)

primary energy carrier of the cell

contains a base, sugar (ribose), and 3 phosphates

3 ways ATP transfers energy

1. hydrolysis-releasing phosphate (Pi)

2. hydrolysis-releasing pyrophosphate (PPi)

3. phosphorylation of organic molecule

3 main routes bacteria and archaea use to catabolize glucose

1. glycolysis (embden-meyerhof-parnas/EMP pathway)

2. enter-doudoroff (ed) pathway

3. pentose phosphate pathway (ppp)/pentose pathway shunt

glycolysis (emp)

conversion of glucose into pyruvate

occurs in the cytoplasm and functions in the absence or presence of oxygen

involves 10 distinct reactions divided into 2 stages

glycolysis stage 1

energy investment

-glucose activated by phosphorylation that convert it to fructose 1,6 BP (2 ATP invested)

-F1,6BP cleaved into two 3-C isomers-dihydoxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P)

hexokinase and phosphofructokinase

2 enzymes in glycolysis that phosphorylates the substrate, using a phosphate group from ATP and producing ADP

aldolase

cleaves fructose 1,6-bisphosphate to produce DHAP and G3P in glycolysis

glycolysis stage 2

energy yield

-2 G3Ps ultimately converted into 2 pyruvates

-redox rxns produce 2 NADH

-4 ATP produced via substrate-level phosphorylation

glycolysis steps

1. glucose activated to glucose 6-phosphate by hexokinase; ATP invested

3. isomerizes to fructose 6-phosphate

4. phosphorylated to fructose 1,6-bisphosphate by phosphofructokinase; ATP invested

5. cleaved to DHAP and G3P by aldolase

6. G3P-> 1,3-bisphosphoglycerate by G3P dehydrogenase; NADH produced

7. phosphorylated to 3-phosphoglycerate + H+ by phosphoglycerate kinase; ATP produced

8. isomerizes to 2-phosphoglycerate + H+

9. dehydrated to phosphoenolpyruvate + H+ by enolase

10. phosphorylated to pyruvate by pyruvate kinase; ATP produxced

glyceraldehyde 3-phosphate dehydrogenase

phosphorylates G3P to 1,3-bisphosphoglycerate and removes 2e-

produces NADH

phosphoglycerate kinase

dephosphorylates 1,3-bisphosphoglycerate to 3-phosphoglycerate + H+

produces ATP

enolase

2-phosphoglycerate to phosphoenolpyruvate (PEP)

produces water

pyruvate kinase

phosphoenolpyruvate to pyruvate

produces ATP

substrate-level phosphorylation

enzyme catalyzed transfer of phosphate from high energy molecule to ADP to produce ATP

requires kinase enzyme

net yield of glycolysis (EMP)

-2 pyruvate

-2 ATP

-2 NADH

net yield of entner-doudoroff pathway (ED)

-2 pyruvates

-1 ATP

-1 NADH

-1 NADPH

key intermediate of glycolysis

G3P

key intermediate of ED pathway

6-P-gluconate

net yield of pentose phosphate pathway (PPP)

-3 7-carbon sugar phosphates

-1 ATP

-2 NADPH

key intermediate of PPP

ribulose 5-P

fermentation

partial breakdown of pyruvate without use of ETS and terminal electron acceptor

-recycles NADH to NAD+

-produces acid and/or ethanol

-mostly doesnt generate ATP

fermentation pathways

homolactic, ethanolic, heterolactic, mixed-acid

homolactic fermentation

produces 2 lactic acids

ethanolic fermentation

produces 2 ethanols and 2 CO2

heterolactic fermentation

produces 1 lactic acid, 1 ethanol, and 1 CO2

mixed-acid fermentation

produces acetate, formate, lactate, succinate, ethanol, H2, and CO2

turns MR red

diagnostic microbiology

use biochemical tests to identify microbe causing disease

tests: phenol red broth, sorbitol macconkey agar, methyl red broth, voges-proskauer

phenol red broth test

turns yellow with production of acid

sorbitol macconkey agar

differentiates pathogenic (white) from nonpathogenic (red) E. coli

red colonies ferment sorbitol while white colonies dont

methy red test

turns red if organism uses mixed acid fermentation to produce acids

voges-proskauer test

tests for organisms that use butylene glycol pathway and produce acetoin

-positive: deep red

-negative: copper color

why organisms use TCA

produces additional metabolites and more NADH and FADH2

pyruvate conversion

catalyzed by pyruvate dehydrogenase complex (PDC) to acetyl coenzyme-A for transport into mitochondria (in eukaryotes)

produces acetyl coA, CO2, NADH, and H+

tricarboxylic acid cycle (TCA, citric acid, krebs)

completes the breakdown of glucose to produce 8 NADH, 6 CO2, 2 ATP, and 2 FADH2 (per glucose)

NADH and FADH2 then bring electrons to ETC

steps of TCA cycle

1. acetyl CoA condenses with 4-C oxaloacetate to form 6-C citrate

2. citrate->isocitrate

3. isocitrate undergoes oxidative decarboxylation to become alpha-ketoglutarate. NADH and CO2 produced

4. ->succinyl-coA producing NADH and CO2

5. ->succinate producing ATP

6. ->fumarate, produce FADH2

7. ->malate, add water

8. ->oxaloacetate, produce NADH

substrates in TCA cycle

oxaloacetate, citrate, isocitrate, alpha-ketoglutarate, succinyl-coA, succinate, fumarate, malate

citrate synthase

couples acetyl-CoA to oxaloacetate, forming citrate and CoA-SH

aconitate hydrase

produces isocitrate

isocitrate dehydrogenase

catalyzes oxidative decarboxylation of isocitrate to alpha-ketoglutarate

produces NADH and CO2